Whole-genome sequencing of alpaca revealed variants in KIT gene potentially associated with the white coat phenotype

The association between variants on TYRP1, TYRP2, MC1R, KITLG, KIT, ASIP and MITF genes and fleece color has been described for alpaca and llama (Zhu et al., 2019; Feeley et al., 2016; Chandramohan et al., 2015; Anello et al., 2022; Pallotti et al., 2020; Shah et al., 2023; Jones et al., 2019; Tan et al., 2022). In this regard, variants on KIT were associated to white and blue-eyed white phenotype in alpaca (Jones et al., 2019; Tan et al., 2022) due to its important role in melanogenesis (Roskoski, 2005). Moreover, previous segregation analysis suggested that the inheritance of white color in alpaca was due to a single gene which is completely dominant over black and brown pigmentation (Valbonesi et al., 2011). In the present study, the segregation of KITLG, KIT and MITF genes was analyzed to find variants associated to white coat phenotype in alpaca. Six Peruvian alpacas belonging to two test-cross families were tested: white huacaya male × pigmented suri female and white suri male x pigmented huacaya female which gave birth to one white suri and one pigmented huacaya, respectively (Table 1). The animals were raised at the experimental station of the INIA (the Peruvian National Institute for Agronomic Innovation) located in Quimsachata, Peru. Sampling of the animals and genomics analysis were performed as reported in Pallotti et al. (2023). Briefly, genomic DNA extracted from skin biopsies underwent whole-genome sequencing on Illumina NovaSeq 6000 System, with a 150x2 bp mode, and an average sequencing depth of 25X. After quality control and alignment to VicPac3 reference genome, the variants were called using the standard joint-call GATK pipeline. All the variants were annotated using SNPeff and filtered according to the segregation of the white coat phenotype. No segregating variants for white coat color were identified in KITLG and MITF. On the contrary, two different KIT variants segregated in white animals (Table 1): two white alpacas, belonging to family 1, were heterozygous (G/A) for the c.35G>A (p.Arg12His) variant, while one white alpaca, belonging to family 2, was heterozygotes (G/C) for c.982G>C (p.Val328Leu) variant. Conversely, all the pigmented animals showed a homozygous G/G genotype. The protein functional domains predicted using InterProScan (Quevillon et al., 2005) suggested that the two mutations were in the N-terminal region of the signal peptide and in the extracellular region which are essential for the binding of the appropriate ligand and consequent activation of KIT (Roskoski, 2005). In addition, such results may suggest a dominant inheritance for alpaca white color as proposed by previous segregation studies (Valbonesi et al., 2011). Although these results must be validated on larger sample, our findings refine the current understanding of the association between gene variants and white color in alpaca and suggest new potential KIT variants as a starting point for further studies on the genetics of white color in alpaca.